Peroxidases and peroxidase conjugates, that is, peroxidases coupled to an immunological component, are relatively unstable, particularly at low concentrations. As a result, compositions containing peroxidases and/or peroxidase conjugates without a suitable stabilizer to inhibit the enzyme inactivation process, exhibit poor shelflife properties, thereby decreasing their commercial applicability.
Peroxidases are enzymes which catalyze the oxidation of certain compounds such as for example, o-phenylene diamine, during which oxidation, a peroxide, in particular hydrogen peroxide, functions as an "acceptor" for a proton donor molecule. Peroxidases may be obtained from plants, for example, horseradish peroxidase (HPO); from vertebrate animals, for example, lactoperoxidase; and from microorganisms such as cytochrome peroxidase from Pseudomonas.
Peroxidases are used for a variety of purposes, in particular, as detectable markers in immunological assay techniques for the detection and determination of immunocomponents, such as haptens, antigens or antibodies. The use of peroxidase labeled immunoreactants is of particular value because the activity or presence of the peroxidase enzyme may be detected visually and the level of activity may be discerned by colorimetric means.
Immunoassay kits useful in performing enzyme immunoassays usually contain as an essential constituent a certain amount of an immunocomponent coupled to a peroxidase. Since such kits will be subject of shipping and storing for variable lengths of time before use, it is essential that the activity of the peroxidase conjugate be maintained as long as possible.
Enzyme conjugates and in particular peroxidase conjugates, are generally stored in an immunological reaction medium containing serum protein, such as for example, fetal calf serum. It has been noted that serum protein contributes to the stability of the conjugate as compared to the stability of the conjugate alone. It is theorized that the serum matrix contributes to maintaining the special structure of the enzyme. However, it has also been observed that the serum protein contributes to the inactivation of the enzyme by extracting detachable hemin moieties from the enzyme structure. This denaturation of peroxidase appears to be due to hemin interactions between the peroxidase and hemin binding proteins from the calf serum. In order to deminish the attraction and thereby the interaction between hemin and serum proteins, various compounds have been proposed as stabilizers for peroxidase compositions. Dawson, et al, in U.S. Pat. No. 4,169,012, describes the use of polyvalent metal ions as stabilizers for peroxidase compositions. Shaffar, in U.S. Pat. No. 4,252,896, discloses the use of 8-anilino-1-naphthalene sulfonic acid (ANS) as a stabilizer for peroxidase compositions.
It is an object of the present invention to provide a novel stable peroxidase composition wherein the peroxidase activity of the composition is maintained for a substantial period of time.